The number of IgG molecules bound to the erythrocyte surface for a given
agglutination score in the antiglobulin test was studied with several different examples of
anti-D, anti-E, anti-c, anti-Kell, anti-Fy^a, anti-Jk^a and immune anti-A antisera. The serological
scores show a significant correlation with the mean values for bound IgG molecules
within a restricted range, although the number bound for a given score may vary by up to
20%. The limit of detection was 100-120 IgG molecules per cell and when over 1,000 were
bound, the cells were completely agglutinated. Anti-Kell bound under low ionic strength
saline conditions required a greater number of molecules for a given agglutination strength.
The relatively low levels of bound IgG necessary to give strong agglutination make the direct
antiglobulin test (DAT) less valuable for following the progress of auto-immune haemolytic
anaemia (AIHA) than a quantitative test. The latter test does not, however, provide any
additional information in AIHA cases with a negative DAT as in these the anaemia does not
appear to be due simply to the number of bound IgG molecules. Detection of certain
antibodies may not be achieved simply by increasing the sensitivity of the antiglobulin test
when correctly performed.
A method is described for the production of an anti-C3+d specific antiserum in
rabbits. The method does not require purification of C3 but relies on the binding of human
C3 to the rabbit erythrocytes following activation of the alternative pathway. The antiserum
produced is suitable for use as either a serological reagent or for immunoprecipitation.
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